The present invention relates to a novel tissue culture system that provides for the long term culture of proliferating hepatocytes that retain hepatic function. Disclosed are methods and compositions for ex vivo culturing of hepatocytes and nonparenchymal cells on a matrix coated with a molecule that promotes cell adhesion, proliferation or survival, in the presence of growth factors, resulting in a long-term culture of proliferating hepatocytes that retain hepatic function. The co-culturing method results in the formation of matrix/hepatic cell clusters that may be mixed with a second structured or scaffold matrix that provides a three-dimensional structural support to form structures analogous to liver tissue counterparts. The hepatic cell culture system can be used to form bio-artificial livers through which a subjects blood is perfused. Alternatively, the novel hepatic cell culture system may be implanted into the body of a recipient host having a hepatic disorder. Such hepatic disorders, include, for example, cirrhosis of the liver, induced hepatitis, chronic hepatitis, primary sclerosing cholangitis and alpha, antitrypsin deficiency.
The present invention is based on the discovery that mixed cultures of proliferating hepatocytes and nonparenchymal cells, grown on a collagen-coated matrix in medium containing hepatocyte growth factor (HGF) and epidermal growth factor (EGF), maintain their capacity to proliferate while retaining hepatic functions.
One of the major functions of the liver is to break down harmful substances absorbed from the intestine or manufactured elsewhere in the body, followed by their excretion as harmless by-products into the bile or blood. Abnormalities of liver function caused by insult to and/or death or malfunction of the cells in the liver can lead to a variety of different hepatic disorders including cirrhosis of the liver or hepatitis. Treatment of such disorders may include whole liver transplants, although this treatment is limited by organ availability, surgical complications, and immunologically-mediated graft rejection normally associated with liver transplantation.
While hepatocyte transplantation has been considered as an alternative to whole-organ transplantation, major technical barriers such as the inability to transfer donor hepatocytes into the liver of a recipient, in numbers to provide a beneficial result, have limited the usefulness of this approach. One of the major difficulties in constructing artificial liver tissue is that, to function effectively, the artificial liver tissue requires functionally active, differentiated hepatocytes present at high densities. Future success with artificial liver tissue will depend on the development of systems in which hepatocytes attached to matrices and packed at high density can retain long term their full functional capacity.
To generate artificial liver tissue, it will be necessary to provide in vitro cultures of hepatocytes. Unfortunately, one of the problems associated with the culturing of hepatocytes is that gene expression deteriorates rapidly as the hepatocytes proliferate. Likewise, long-term cultures of hepatocytes having stable gene expression can only be maintained in the absence of cell proliferation. Thus, one of the long-standing goals of culturing hepatocytes is the establishment of proliferating cultures with long-term gene expression.
A number of culture techniques have been developed that permit primary hepatocyte cultures to grow and/or express complex patterns of hepatocyte differentiation (Mitaka, et al., 1995, Biochem Biophys Res Commun 214: 310-317; Cable, 1997, Hepatology 26: 1444-1445; Block, et al., 1996, J. Cell Biol. 132: 1133-1149). Conditions have also been established that allow mature hepatocytes to enter into clonal expansion in cell culture (Block, et al., 1996, J. Cell Biol. 132: 1133-1149). For example, hepatocytes cultured in chemically defined hepatocyte growth medium (HGM) enter into DNA synthesis in response to polypeptide mitogens, notably epidermal growth factor (EGF), transforming growth factor-xcex1 (TGF-xcex1), and hepatocyte growth factor (HGF). These mitogens induce multiple rounds of DNA synthesis and expansion of the cell population. The proliferating cells, however, lose most markers of hepatocyte differentiation while they retain expression of hepatocyte associated transcription factors HNF1, HNF4, and HNF3. In addition, proliferation of adult hepatocytes has been observed in serum-free medium supplemented with nicotinarnide and epidermal growth factor (EGF) (Mitaka, T., et al., 1991, Hepatology 12: 21-30; Mitaka, T., et al., 1992, Hepatology 10:440-447; Mitaka, T., et al., 1993, J. Cell Physiol, 147: 461-468; Mitaka, T., et al., Cancer Res, 1993, 53: 3145-3148; Block, G. D., et al., 1996, J. Cell Biol. 132:1133-1149; Tateno, C., et al., 1996, Am J. Pathol 148: 383-392).
Previous studies have indicated that a fundamental parameter that best determines hepatocyte gene expression in culture is the surrounding matrix. Hepatocytes embedded in complex matrices, such as Matrigel or type I collagen gels, maintain stable phenotypic expression, however, at the expense of cell proliferation. Recently, Mitaka, T. et al. (1999, Hepatology 29: 111-125) showed that small hepatocytes could differentiate to mature hepatocytes that interact with hepatic nonparenchymal cells and extracellular matrix. The mature hepatocytes reconstructed three-dimensional structures, expressed proteins known to be expressed in highly differentiated hepatocytes and the cells survived for more than 3 months, while maintaining hepatic differentiated functions. In addition, Landry et al. (1985, J. Cell Biol. 101:914-923) demonstrated the reconstruction of a three-dimensional cyto-architecture consisting of differentiated hepatocytes, bile duct-like cells and deposited extracellular matrix by the use of spheroidal aggregate culture of hepatic cells isolated from newborn rats. Three-dimensional cell culture systems have also been disordered in which hepatocytes are grown on a pre-established stromal tissue (U.S. Pat. No. 5,624,840). Attempts have also been made to grow a three-dimensional hepatic organoid using a co-culture of hepatocytes and fibroblasts (Senoo, et al., 1989, Cell Biol. Internat. Reports 13:197-206; Takezawa, et al., 1992, J Cell Sci 101:495-501).
A number of devices which perform the function of the liver and involve blood perfusion have been described (Hagger et al., 1983, ASAIO J. 6:26-35; U.S. Pat. No. 5,043,260; U.S. Pat. No, 5,270,192: Demetriou et al., 1986, Ann. Surg 9:259-271). However, a number of problems are associated with the use of such devices for treatment of patients suffering from hepatic failure or dysfunction. Perhaps, the most significant problem is the inability to culture hepatocytes that retain hepatic function for prolonged periods of time, although, attempts have been made to circumvent this problem through the use of transformed hepatocytes that are capable of proliferating indefinitely (U.S. Pat. No. 4,853,324).
Development of a stable support system that would maintain hepatic functions and be useful in stabilizing patients in partial or complete hepatic failure has been a long-term scientific goal in the field of hepatology. Similar devices have revolutionized the treatment of patients with kidney failure and have allowed long-term stabilization of a large population of patients. Currently the use of such devices in treatment of liver failure is quite limited and existing devices are based on rapidly assembled hepatocyte support systems which partially sustain the patient over a very limited period of time, i.e, 24 to 48 hours with declining function over more prolonged term use.
The present invention relates to a novel tissue culture system that provides for long term culture of proliferating hepatocytes that retain their capacity to express hepatic function. The invention generally relates to compositions and methods for generating long term cultures of hepatocytes that can be used to produce three-dimensional hepatic cell culture systems. Such hepatic cell culture systems can be used to form bio-artificial livers that function as perfusion devices. Alternatively, the three-dimensional hepatic cell cultures may be implanted into a subject having a liver disorder.
The method of the present invention comprises the co-culturing of hepatocytes and nonparenchymal cells in the presence of growth factors and a matrix material coated with at least one biologically active molecule that promotes cell adhesion, proliferation or survival. The co-culturing method results in the formation of matrix/hepatic cell clusters containing a mixture of replicating hepatocytes and nonparenchymal cells. The method of the present invention may further comprise the mixing of the matrix/hepatic cell clusters in combination with a second structured, or scaffold matrix, that provides a three-dimensional structural support to form structures analogous to liver tissue counterparts.
Compositions of the present invention include populations of matrix/hepatic cell clusters comprising co-cultures of hepatocytes and nonparenchymal cells bound to a matrix coated with at least one biologically active molecule that promotes cell adhesion, proliferation or survival. Further, the invention provides a three-dimensional hepatic cell matrix system comprising a three-dimensional support matrix containing a population of matrix/hepatic cell clusters comprising hepatocytes and nonparenchymal cells bound to a matrix coated with at least one biologically active molecule that promotes cell adhesion, proliferation or survival.
The compositions of the present invention may be used to form bio-artificial livers through which a host""s blood is perfused. Alternatively, the three-dimensional hepatic cell matrix system may be transplanted to a recipient host for providing hepatic function in subjects with liver disorders. The three-dimensional matrix system is administered in an effective amount to provide restoration of liver function, thereby alleviating the symptoms associated with liver disorders. The present invention, by enabling methods for generating long-term cultures of hepatocytes, provides a safer alternative to whole liver transplantation in subjects having liver disorders including, but not limited to, cirrhosis of the liver, alcohol induced hepatitis, chronic hepatitis, primary sclerosing cholangitis and alpha,-antitrypsin deficiency.
FIGS. 1A-B. Thin sections of cells on beads in roller bottle cultures at day 15 after isolation, stained with toluidine blue.
FIG. 1A. The bead is seen as a hollow space in the center of the cell cluster. Gray material around the bead represents dense type-1 collagen deposition. The collagen surrounds and embeds connective-tissue derived nonparenchymal cells. Cells with hepatocyte morphology surround the connective tissue core.
FIG. 1B. The epithelial cells with hepatocyte morphology form an eccentric growth over a foundation of connective tissue cells. Note the formation of multiple microvilli over the hepatocytes present on the surface.
FIG. 2. Matrix deposition in Stage 1 roller bottle cultures. Panels A, B, and C show depositions of collagen types I, III, and IV, respectively. Collagen types I and III are deposited as broad bands surrounding the beads. Collagen type IV often formed basement membrane structures surrounding hepatocytes arranged in acinar or ductal configurations. Matrix is stained red whereas nuclei of the adjacent cells are stained blue. Visualization was by immunofluorescence microscopy.
FIGS. 3A-C. Electron microscopy of cultures at Stage 1 (Roller bottle).
FIG. 3A. Low magnification view of hepatocytes growing on beads, before addition of Matrigel. Hepatocytes form a continuous multilayer or monolayer culture around the beads and display circuitous, interdigitated cell-cell contacts within the abluminal membrane. Canalicular structures (CC) and tight junctions (TJ) are seen. A 1-micron thick layer of fibrillar collagen (Col) is evident between the hepatocytes"" abluminal membranes and the polystyrene bead. A nonparenchymal cell (NPC) is also seen within the fibrillar collagen layer. Bar=1 mmol/L.
FIG. 3B. Another view of the cytoplasmic features of hepatocytes at stage 1 (Magnification, 4,000xc3x97). Sinusoidal endothelial cells (SEC) are forming a layer of fenestrated endothelium. Fibrillar collagen (Col) and multiple microvilli are seen under the endothelial layer, with a morphology similar to that seen in the space of Disse.
Glycogen (Gly) and lamellae of rough endoplasmic reticulum (RER) are seen in the cytoplasm of the adjacent hepatocytes.
FIG. 3C. Higher magnification of B (10,000xc3x97) showing the fenestrae of the endothelial layer. Collagen fibrils are seen in the interrupted cytoplasmic continuity of the endothelial cell at the site of the formation of the fenestra.
FIGS. 4A-C. Stains for macrophages, endothelial cells, and desmin-positive cells in Stage 1 roller bottle cultures. Visualization by differential interference microscopy. Positive immunohistochemistry is shown as red (complete arrows) whereas nuclei of cells are stained blue (truncated arrows).
FIG. 4A. Macrophages staining positive for ED-1 antigen. Note the xe2x80x9cfoamyxe2x80x9d cytoplasm characteristic of macrophages in some of the cells.
FIG. 4B. Desmin-positive cells.
FIG. 4C. Structures of endothelial cells staining positive for 1CAM1 antigen. One of the endothelial cells contains a nucleus at the field of the image (complete arrow).
FIGS. 5A-B. Migration of cell populations from bead clusters after placement in Matrigel (Collaborative Biomedical, Mass.). Phase contrast microscopy.
FIG. 5A. Nonparenchymal cells (NP) migrate first and spread by attaching to the substratum. Occasional buddings of epithelial cells are seen at a higher focus plane (Hep). Some (arrow) appear to contain a duct. Culture at 1 week in Matrigel. Magnification, 200xc3x97.
FIG. 5B. Multiple buddings of epithelial cells migrate out of the bead clusters at different planes and in all directions. Culture at 20 days in Matrigel. Magnification, 200xc3x97.
FIG. 6. Histology of the epithelial cell buddings in Matrigel at Stage 2 cultures at day 20 in Matrigel. Epithelial cells with hepatocyte morphology (see FIG. 8) are surrounding the central bead core and are arranged in sheets and ducts. Connective tissue deposition is also present underlying the epithelial cell structures. Hematoxylin eosin stain. Magnification, 200xc3x97.
FIG. 7A. Low power electron micrograph of an acinar structure formed from the bead cluster. Evident are the duct-like canalicular structures (C) in the center of the acinar structure. Cells contain extensive RER and numerous mitochondria. A thick, but less electron dense layer of extracellular matrix than that observed for the pre-Matrigel bead is seen between the hepatocytes and the bead, with several fibroblastic (F) type cells residing in the matrix. Barxe2x88x922 mm.
FIG. 7B. High power micrograph of the canalicular structure seen in A. Readily obvious are three extensive tight junctional areas (TJ), desmosomes, RER, Golgi elements, and Mt, mitochondria. Bar=500 nm.
FIG. 8. Formation of plates by hepatocytes at Day 20 in Matrigel. Notice the prominent canalicular network (bright canals, arrows) along the middle of the plate.
FIG. 9. Cellular and matrix immunohistochemistry in Stage 2 cultures in Matrigel. Staining by immunoperoxidase. Panels A,B,C, and D show stains for desmin, Collagen types I, III, and IV, respectively. Desmin-positive stellate cells are interspersed in close proximity to the hepatocytes. Collagen type III shows the strongest immunohistochemical response. Collagen type IV often formed basement membrane structures surrounding hepatocytes arranged in acinar or ductal configurations (arrow).
FIG. 10A Phase contrast microscopy of monolayers developing at 2 to 3 months in Matrigel (Stage 3 cultures) in the presence of HGF and EGF. Magnification 100xc3x97.
FIG. 10B. Magnification 200xc3x97. Notice the extensive canalicular network (bright lines ramifying with short branches along the hepatocyte plates), the pseudo-sinusoidal spaces (S), and the duct-like structures (D).
FIG. 11A. A low power (2,000xc3x97) electron micrograph of hepatocytes in Stage 3 cultures. Notice the longitudinal section of the extensive canalicular network (with microvilli and desmosomes) surrounding the individual hepatocytes.
FIG. 11B. Higher power view (10,000xc3x97) showing detailed cytoplasmic features. Rough endoplasmic reticulum, mitochondria, and Golgi network elements are seen in the individual hepatocytes.
FIG. 12. Expression of several genes in hepatocytes immediately after isolation (Time zero), cells in roller bottle at day 13, cells in roller bottle at day 25, cells in Matrigel (Collaborative Research) cultures at day 25 (12 days after placement in Matrigel at Day 13), and nonparenchymal hepatic cell fraction (5% nonparenchymal hepatocyte contamination) immediately after isolation. Expression of GAPDH is used as a normalizing parameter.
FIGS. 13A-C. Induction of the cytochrome P450 species CYP3A (FIG. 13A), CYP1A (FIG. 13B) and CYP2B1/2 (FIG. 13C) by their characteristic inducers in day 35 cultures. The increase in actual is demonstrated by western immunoblot. C stands for control. Dex (dexamethasone); 3MC (3xe2x80x2 Methylcholanthrene); PB (Phenobarbital) were the inducers used correspondingly.
FIG. 14. Enzymatic Activities. The activities of testosterone 6xcex2-hydroxylase (CYP3A dependent) and ethoxyresorufin O-deethylase (CYP1A dependent) were also measured in the same cultures. As demonstrated, more than 20-fold induction was seen in both cases by the characteristic inducers.